The present invention provides for a radioligand compound that is a useful agent to screen for potential Class III antiarrhythmic compounds and method of screening for this IKr activity using this radioligand compound. In the past, a similar binding assay was conducted using a [3H]-radioligand capable of binding to ether-a-go-go-related gene (ERG) in guinea pig myocytes or patch clamping on intact cells were carried out. See Geonzon, R. et al. J. Mol. Cell Cardiol. 30, p.1691-1701 (1998); Chadwick, C. C. et al. Circulation Research 72, p.707-714 (1993); Duff, H. J. et al. Circulation Research 77, p.718-725 (1995); and Fiset, C. et al. J. Mol. Cell Cardiol. 28, p.185-1096 (1996).
The present invention offers several advantages over the previous methods. Use of a [35S]-labeled radioligand offers the advantage that [35S] is a higher energy and specific activity radioisotope than [3H], allowing for scientists to refine the assay conditions with the use of less radioligand and human ERG (also referred to as hERG) channel, as well as the ability to set up a more sensitive assay. The current binding assay has also been developed using membranes from cells expressing hERG, rather than ERG in intact animal cells, as the source of the ion channel. Thus, the use of human ERG in the present assay will provide investigators with a more relevant binding assay over the animal ERG.
Additionally, membranes are a more convenient source of the ion channel compared to intact cells. A larger throughput is possible as a large amount of the membrane can be made in advance, stored frozen and thawed on the day of the assay. Also, a membrane source of ion channel has both its extracellular and intracellular sites exposed and is not dependent on changes in the physiology of an intact cell for all potential binding sites to be exposed. A final advantage of the [35S]-based binding assay is that it can be carried out with much higher throughput compared to a whole cell patch clamp assay. Thus, the current invention is more suited to broad-based screening. This assay is used as a counterscreen in research programs where long QT activity is not desirable.
U.S. Pat. No. 5,633,247 discloses and claims the unlabelled compound (also referred to as the cold ligand), N-[1′-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydoxyspiro[2H-1-benzopyran-2,4′-piperidin]-6-yl]-methanesulfonamide.